Interaction of the integrin receptors with ligands determines the molecular basis of integrin-dependent cell adhesion. Integrin ligands are typically large proteins with relatively low binding affinities. This makes direct ligand-binding kinetic measurements somewhat difficult. Here we examine several real-time methods, aimed to overcome these experimental limitations and to distinguish the regulation of integrin conformation and affinity. This chapter includes: the use of a small ligand-mimetic probe for studies of inside-out regulation of integrin affinity and unbending, real-time cell aggregation and disaggregation kinetics to probe integrin conformational states and the number of integrin–ligand bonds, as well as the real-time monitoring of ligand-induced epitopes under signaling through G-protein-coupled receptors, and others. Experimental data obtained using these novel methods are summarized in terms of the current model of integrin activation.
